Fecal PCR test for Tritrichomonas foetus

General information

Tritrichomonas foetus is an important cause of diarrhea
in cats. This assay amplifies the DNA of T. foetus in
feces and has been shown to detect as few as 10 organisms per 1
gram of feces. All PCR reactions are performed with positive and
negative controls and a restriction enzyme digest is performed on
positive samples to ensure the amplified DNA sequence is specific
to T. foetus.

For this test we will require up to 1 gram of fresh feces. To
increase the probability of detecting infection, the cat should be
sampled when it is having diarrhea. The fecal sample can be
collected from a spontaneously voided stool, using a fecal loop, or
using the colonic saline flush technique (see below). The sample
must be free from cat litter as some components of certain cat
litters may inhibit PCR reactions. Collect the fecal sample as
promptly as possible. However, once the fecal sample has been
collected it can be stored in a refrigerator for up to one week
prior to shipping. Please ship cooled samples overnight with a gel
ice pack (the samples do not have to be frozen but these
precautions will help prevent the samples getting overheated during
transit). The turnaround time for this assay is 1-2 business days
after receipt of the sample at the Gastrointestinal Laboratory.

General information about Tritrichomonas foetus

When evaluating feline patients with diarrhea it is essential to
include infectious etiologies on the list of differential
diagnoses. One such infectious organism is Tritrichomonas
foetus, a flagellated protozoal parasite that is usually
associated with venereal trichomoniasis in cattle. Recently, T.
foetus has also been identified as an intestinal pathogen in
cats. Diarrhea has been reported in cats after both experimental
and natural infection. Although the true prevalence of T.
foetus infection in cats is unknown, it is suspected to be
relatively high. In one study, 31% of 117 cats examined at an
international cat show were infected with T. foetus.

While cats of any age, breed, or sex can be infected, young cats
that are densely housed (e.g., cats in catteries, animal shelters,
or multi-cat households) seem to be at increased risk. Infection is
most commonly seen in young cats (i.e., those that are less than 12
months of age), but older cats may also be infected. T.
foetus primarily colonizes the surface of the colonic mucosa,
leading to chronic large bowel diarrhea. Without appropriate
treatment cats usually remain persistently infected. While diarrhea
may spontaneously resolve, cats will often experience recurrent
bouts of diarrhea after being exposed to stress. Cats infected with
T. foetus generally appear healthy but show an increased
frequency of defecation with loose to liquid stools, which may
contain blood and/or mucus. Fecal incontinence is also frequently
observed. The anal region often appears edematous and may become
painful with severe diarrhea. A rectal prolapse may occur in some
cases.

Who should be tested?

In general, kittens and young cats from multi-cat environments
with chronic diarrhea should be tested for T. foetus
infection. However, it should be noted that cats in single cat
households and older cats can also get the disease, so cats with
clinical signs that do not have another obvious cause for their
diarrhea should also be tested. Furthermore, cats that are
unresponsive to treatment for suspected Giardia spp. infection
should be tested, as the two parasites are sometimes confused on
routine fecal smear evaluation.

Diagnosis

Diagnosis of a T. foetus infection can be made by
identification of tritrichomonads on a direct fecal smear
examination, fecal culture, PCR analysis of fecal material, or by
colonic mucosal biopsy. Disadvantages of direct fecal smear
examination include a low sensitivity (14%), low specificity
(T. foetus can be misdiagnosed as Giardia spp. or the
nonpathogenic Pentatrichomonas hominis), and the fact that only
fresh fecal samples can be used.

Tritrichomonas foetus can also be cultured in-house
using the commercially available culture system In Pouch™ TF
(Biomed Diagnostics, San Jose, CA). Pouches should be inoculated
with less than 0.1 g of freshly voided feces and then incubated at
25°C. Pouches need to be evaluated under a microscope every couple
of days. Results are usually obtained between 1 and 11 days of
setting up a pouch. Although fecal culture is more sensitive than
direct fecal smear examination, difficulties in interpretation of
results, the necessity of using freshly voided feces, and the fact
that results might not be available for up to 11 days, are
important disadvantages of this method.

Tritrichomonas foetus DNA can be amplified from fecal
samples by PCR. PCR has been shown to be the most sensitive method
for detecting T. foetus in fecal samples, and is ideally
suited for direct diagnosis of T. foetus infection.
Advantages of PCR testing compared to culture include a higher
sensitivity, faster turnaround time, and easier handling and
storing of samples because DNA is relatively stable under various
temperature conditions. Using the colonic saline flush technique
(http://www.youtube.com/watch?v=JMfZ9M80V8E) or a fecal loop to
obtain a fecal sample directly may help increase the probability of
detecting T. foetus. The sediment of the sample of fluid
obtained from a colonic saline flush should be separated by
centrifugation or allowing it to settle. This material is then
submitted for PCR. Ideally, fecal samples should be collected when
the cat is having diarrhea.

Therapy

Until recently, a successful treatment strategy for T.
foetus infections in cats was not available. However, studies
have shown that administration of ronidazole may be effective in
both resolving diarrhea and eradicating T. foetus. Based
on a study of the pharmacokinetics of ronidazole in cats the
current treatment recommendation is a dose of 30 mg/kg by mouth
once daily for 14 days. Retesting may be performed 2 weeks after
the last dose of ronidazole. Apparent treatment failure may be due
to lack of patient or owner compliance, administration of an
inappropriate dose or duration of ronidazole, reinfection from the
environment or from other cats, or infection with a ronidazole
resistant strain of T. foetus. Ronidazole can cause
neurotoxicosis in cats so patients should be monitored carefully
during treatment and treatment should be discontinued if any
neurological or other clinical signs develop. Some infected cats do
not have diarrhea so testing non-diarrheic cats in the same
household is warranted. Where possible, in multi-cat households
separation of infected and non-infected cats is advised. Infection
is via the fecal-oral route and environmental contamination may be
important in the epidemiology of T. foetus. A recent study
documented trophozoite survival in water, urine, and cat food but
not cat litter. Further information on T. foetus for cat
owners is available from Dr. Jody Gookin's website (
http://www.cvm.ncsu.edu/docs/personnel/gookin_jody.html ).